miRNeasy Serum/Plasma Kit

For purification of cell-free total RNA, including miRNA, from animal and human plasma and serum

Internal control available for normalization

Minimal elution volume

High-purity RNA suitable for all downstream applications

Easy, robust procedures

Automatable protocol

The miRNeasy Serum/Plasma Kit is designed for purification of cell-free total RNA — primarily miRNA and other small RNA — from small volumes of serum and plasma. RNA from serum and plasma typically consists of molecules <100 nucleotides. The miRNeasy Serum/Plasma Kit includes a miScript Primer Assay that detects the miRNeasy Serum/Plasma Spike-In Control, providing a convenient system for normalization. The miRNeasy Serum/Plasma Spike-In Control (ordered separately) is a C. elegans miR-39 miRNA mimic and is supplied lyophilized at 10 pmol per tube. Purification can be automated on theQIAcube.

The miRNeasy Serum/Plasma Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Performance

The miRNeasy Serum/Plasma Kit efficiently purifies RNA from up to 200 µl serum or plasma. RNA prepared using miRNeasy Kits is highly pure and ready for use in sensitive downstream applications. miRNeasy procedures minimize the possibility of contamination with salts or phenol, which could interfere with later analyses.

Principle

One of the most exciting areas of current miRNA research involves the assessment of miRNAs present in serum or plasma samples. The presence of relatively stable, extracellular miRNAs in serum and plasma has generated great interest in the potential use of changes in these miRNA levels as noninvasive biomarkers for a variety of diseases. Using RNA purified from serum or plasma, unique expression of several miRNAs has been shown for some cancers. The miRNeasy Serum/Plasma Kit enables RNA purification from small volumes of plasma or serum. RNA from serum and plasma typically consists of molecules <100 nucleotides. Purified RNA can then be used for biomarker discovery with the miScript PCR System.

The miRNeasy Serum/Plasma Kit combines phenol/guanidine-based lysis of samples and silica-membrane-based purification of total RNA. QIAzol Lysis Reagent, included in the kit, is a monophasic solution of phenol and guanidine thiocyanate, designed to facilitate lysis of tissues, to inhibit RNases, and also to remove most of the cellular DNA and proteins from the lysate by organic extraction.

Use of an internal, normalization control

There is currently no clear consensus in the research community on what should be used as an internal normalization control for miRNA expression profiling in a serum or plasma sample. Many researchers choose to spike a synthetic miRNA into their RNA prep to help monitor RNA recovery and reverse transcription efficiency. QIAGEN recommends the miRNeasy Serum/Plasma Spike-In Control (ordered separately) for use as an internal control for miRNA expression profiling in serum or plasma. The miRNeasy Serum/Plasma Spike-In Control is a C. elegans miR-39 miRNA mimic that can be easily detected by real-time RT-PCR using the miScript PCR System in combination with the Ce_miR-39_1 miScript Primer Assay (provided in the miRNeasy Serum/Plasma Kit). For more details, consult the miRNeasy Serum/Plasma Handbook.

Procedure

Serum or plasma samples are lyzed in QIAzol Lysis Reagent. After addition of chloroform, the lysate is separated into aqueous and organic phases by centrifugation. RNA partitions to the upper, aqueous phase, while DNA partitions to the interphase and proteins to the lower, organic phase or the interphase.

The upper, aqueous phase is extracted, and ethanol is added to provide appropriate binding conditions for all RNA molecules from approximately 18 nucleotides upwards. The sample is then applied to the RNeasy MinElute spin column, where the total RNA binds to the membrane and phenol and other contaminants are efficiently washed away. High-quality RNA is then eluted in a small volume of RNase-free water.

Serum and plasma contain primarily small RNAs, therefore separate purification of small and large RNA fractions is not necessary.

Normalization using the miRNeasy Serum/Plasma Spike-In Control

During miRNA purification using the miRNeasy Serum/Plasma Kit, the miRNeasy Serum/Plasma Spike-In Control is added to samples after the addition of QIAzol Lysis Reagent, prior to addition of chloroform and phase separation. After real-time RT-PCR, the CT value obtained with the assay targeting the synthetic miRNA permits normalization between samples, which can control for varying RNA isolation yields and amplification efficiency. In addition, RNA recovery can be assessed by comparing the CT value with a standard curve of the synthetic miRNA generated independently of the RNA isolation procedure.

Automated miRNA purification on the QIAcube

Purification of total RNA, including miRNA, from the aqueous phase after phase separation can be automated on the QIAcube. The innovative QIAcube uses advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into your laboratory workflow. Sample preparation using the QIAcube follows the same steps as the manual procedure (i.e., lyse, bind, wash, and elute), enabling you to continue using the miRNeasy Serum/Plasma Kit for purification of high-quality RNA.

Applications

The miRNeasy Serum/Plasma Kit allows purification of cell-free total RNA, including miRNA, for use in a variety of applications, including: